Activation of phosphatidylinositol 3-kinase, protein kinase B, and p70 S6 kinases in lipopolysaccharide-stimulated raw 264.7 cells: Differential effects of rapamycin, Ly294002, and Wortmannin on nitric oxide production

Phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B are critic al players in cell proliferation and survival. Their downstream effector pr otein kinase, p70 S6 kinase, has an established role in protein translation . The mechanism by which bacterial LPS induces production of nitric oxide ( NO) in murine macrophages is incompletely understood, and a role for PI 3-k inase/p70 S6 kinase pathway had not been previously investigated. In this s tudy we demonstrate that LPS induced a fivefold activation of p70 S6 kinase and a twofold stimulation of PI 3-kinase. Pretreatment of Raw 264.7 cells with either rapamycin or Ly290042 completely blocked LPS-induced activation of p70 S6 kinase. Protein kinase B was also activated (twofold) by LPS and was only minimally affected by these inhibitors. PI 3-kinase activity was inhibited by both Ly294002 and wortmannin, The effects on NO production by these agents were strikingly different, While both rapamycin and Ly294002 r esulted in almost complete inhibition of NO production, wortmannin was inef fective. Surprisingly, none of the inhibitors reduced the production of the inducible nitric oxide synthase protein (iNOS) as determined by immunoprec ipitation, In vivo labeling studies revealed that the iNOS protein was phos phorylated in concordance with the production of NO. We conclude that LPS-m ediated NO production occurs via a PI 3-kinase-independent, but FKBP12-rapa mycin-associated protein-dependent, pathway in RAW cells by a mechanism pro bably involving phosphorylation of iNOS.